Design, synthesis and biological evaluation of dipeptides as novel non-covalent 20S proteasome inhibitors

Article abstract of DOI:10.1080/10286020.2021.1910241

Based on the interaction modes of the natural 20S proteasome inhibitors TMC-95A, we have previously discovered a dipeptide 1. To explore the SAR around compound 1, we designed and synthesized a series of dipeptides (8–38) with a fragment-based strategy. Among them, nine compounds showed significant inhibitory activities against the chymotrypsin-like activity of human 20S proteasome with IC₅₀ values at the submicromolar level, which were comparable or even superior to the parent compound 1. Meanwhile, they displayed no significant inhibition against trypsin-like and caspase-like activities of 20S proteasome. The results suggested the feasibility to design dipeptides as novel and potent 20S proteasome inhibitors. (Figure presented.).

Full text of DOI:10.1080/10286020.2021.1910241

Journal of Asian Natural Products Research
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Design, synthesis and biological evaluation of
dipeptides as novel non-covalent 20S proteasome
inhibitors
Ya-Jun Yang, Ke Wang, Ying Yang, Fang-Fang Lai, Xiao-Guang Chen & Zhi-Yan
Xiao
To cite this article: Ya-Jun Yang, Ke Wang, Ying Yang, Fang-Fang Lai, Xiao-Guang Chen & Zhi-
Yan Xiao (2021) Design, synthesis and biological evaluation of dipeptides as novel non-covalent
20S proteasome inhibitors, Journal of Asian Natural Products Research, 23:5, 436-451, DOI:
10.1080/10286020.2021.1910241
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
2021, VOL. 23, NO. 5, 436–451
https://doi.org/10.1080/10286020.2021.1910241
Design, synthesis and biological evaluation of dipeptides
as novel non-covalent 20S proteasome inhibitors
Ya-Jun Yanga, Ke Wanga, Ying Yanga, Fang-Fang Laib, Xiao-Guang Chenb and
Zhi-Yan Xiaoa
aBeijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
b
100050, China; The State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Material Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, China
ABSTRACT
ARTICLE HISTORY
Received 22 January 2021
Accepted 25 March 2021
Based on the interaction modes of the natural 20S proteasome
inhibitors TMC-95A, we have previously discovered a dipeptide 1.
To explore the SAR around compound 1, we designed and syn-
thesized a series of dipeptides (8–38) with a fragment-based strat-
egy. Among them, nine compounds showed significant inhibitory
activities against the chymotrypsin-like activity of human 20S pro-
teasome with IC50 values at the submicromolar level, which were
comparable or even superior to the parent compound 1.
Meanwhile, they displayed no significant inhibition against tryp-
sin-like and caspase-like activities of 20S proteasome. The results
suggested the feasibility to design dipeptides as novel and potent
20S proteasome inhibitors.
KEYWORDS
20S proteasome inhibitors;
non-covalent; dipeptides
1. Introduction
The ubiquitin-proteasome system (UPS) is a proteolytic system responsible for the
precise control of cellular protein homeostasis, and it participates in a great variety of
biological processes [1, 2]. As the main component of UPS, the 26S proteasome is a
CONTACT Zhi-Yan Xiao
xiaoz@imm.ac.cn
Beijing Key Laboratory of Active Substance Discovery and
Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
437
Figure 1. The structures of TMC-95A and compound 1.
proteolytic machine with multiple functions. In eukaryotes, the 26S proteasome is
composed of the regulatory 19S and the catalytic 20S particles [3]. The cylinder-
shaped 20S proteasome is the catalytic core of 26S proteasome and is formed by four
stacked rings with twenty-eight subunits [4]. Among them, only b1, b2 and b5 subu-
nits are proteolytically active, which are associated with the caspase-like (C-L), tryp-
sin-like (T-L) and chymotrypsin-like (CT-L) activities, respectively [5, 6]. The
approval of the 20S proteasome inhibitors, bortezomib, carfilzomib and ixazomib, has
validated 20S proteasome as a therapeutic target for cancer treatment [7]. However,
as covalent and peptidic inhibitors, their clinical application is hindered by issues of
severe side effects, acquired drug resistance, and unsatisfactory pharmacokinetic pro-
files [8–10]. Non-covalent proteasome inhibitors are generally assumed to have poten-
tial benefits over their covalent peers [11–12]. Therefore, the development of novel
non-covalent 20S proteasome inhibitors has drawn extensive research interests in
recent years.
TMC-95A (Figure 1) is a potent non-covalent 20S proteasome inhibitor isolated
from the fermentation broth of the ascomycete fungus Apiospora montagnei [13].
Although the complex structure of TMC-95A impedes a fully SAR exploration of the
chemical space around it, some simplified mimics, including cyclic tripeptides
[14–16], linear tripeptides and dipeptides [17–19], were rationally designed based on
its binding mode [20]. Inspired by the structure of TMC-95A, we designed a series of

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Y.-J. YANG ET AL.
Scheme 1. Synthetic route of compounds 8–38. Reagents and conditions: (a) HATU, DIEA, DMF
(N,N-dimethyllformamide), rt, overnight; (b) 10% Pd/C, MeOH (methanol), H2, rt, overnight; (c) cor-
responding amines, HATU, DIEA, DMF, rt, overnight;(d)TFA (trifluoroacetic acid), DCM (dichlorome-
thane), rt, 4 h; (e) corresponding acids, HATU, DIEA, DMF, rt, overnight.
linear dipeptides previously, and compound 1 (Figure 1) showed apparent inhibition
against the CT-L activity of human 20S proteasome with an IC50 value of 0.66 lM.
As revealed by molecular docking, molecular areas of P4, P3 and P1 in compound 1
protrude into the S4, S3 and S1 pockets, respectively, and the dipeptide backbone
could form extensive hydrogen bonds with the key residues of the active site [21].
The results encouraged us to further develop more potent linear dipeptides as novel
non-covalent 20S proteasome inhibitors. Based on a fragment-based strategy, we
approach the optimization of compound 1 by a stepwise evolution of the P4, P3 and
P1 moieties to explore the SAR of the chemical space around compound 1, and hope-
fully to further improve the activity profile of this compound class.
2. Results and discussion
2.1. Chemistry
The synthetic route of target compounds was shown in Scheme 1. First, the
corresponding Boc-protected amino acids 2 and H-L-Val-OBn 3 were coupled in the
presence of HATU (2-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexa-
fluorophosphate) and DIEA (N,N-diisopropylethylamine) to give dipeptides 4. The
dipeptides 4 were subjected to hydrogenation using 10% Pd/C to liberate the carbox-
ylic group and produce acids 5. Then acids 5 were coupled with corresponding
amines to afford compounds 6, which were treated with trifluoroacetic acid to free
the amino group and give compounds 7. Finally, compounds 7 were coupled with
corresponding acids to provide target compounds 8–38.
2.2. Biological evaluation
The inhibitory profiles of the synthesized dipeptides were initially assayed against the
CT-L activity of human 20S proteasome as described previously [14]. As shown in
Table 1, most compounds displayed moderate to potent inhibitory activities, and nine
of them showed IC50 values at the submicromolar level, which are comparable or
even superior to the parent compound 1.

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439
Table 1. Inhibitory activities of compounds 8–38 against CT-L, T-L and C-L activities of human
20S proteasome.
IC50 (lM)
Comp.
1
R1
R2
R3
CT-L
0.66
T-L
ND
C-L
ND
8
5.10
17.2
1.40
ND
ND
ND
ND
ND
ND
9
10
11
12
0.84
0.17
>100
>100
>100
>100
13
0.53
>100
>100
14
15
5.10
2.06
ND
ND
ND
ND
16
17
0.57
0.87
>100
>100
>100
>100
18
0.20
>100
>100
(continued)

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Y.-J. YANG ET AL.
Table 1. Continued.
IC50 (lM)
Comp.
19
R1
R2
R3
CT-L
4.50
T-L
ND#
C-L
ND
20
0.89
>100
>100
21
22
3.50
3.70
ND
ND
ND
ND
23
24
25
11.8
5.50
0.89
ND
ND
ND
ND
>100
>100
26
27
28
29
9.06
5.68
3.54
6.90
ND
ND
ND
ND
ND
ND
ND
ND
30
31
1.65
2.98
ND
ND
ND
ND
(continued)

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441
Table 1. Continued.
IC50 (lM)
Comp.
32
R1
R2
R3
CT-L
0.36
T-L
C-L
>100
>100
33
34
5.70
15.4
ND
ND
ND
ND
ꢀ
35
NA
ND
ND
36
37
NA
NA
ND
ND
ND
ND
38
NA
ND
ND
ꢀ
NA: The percentage inhibition was less than 50% under the concentration of 10 lM.
#ND: not determined.
Preliminary SAR could be deduced from Table 1. For compounds 8-22, we have
the P1 and P3 moieties of compound 1 retained to preserve the interactions with S1
and S3 pockets, while have its P4 group replaced with various hydrophobic substitu-
ents to explore SAR in this molecular area. The introduction of phenyl or naphthyl
groups (8-10) led to an apparent drop of activity. Groups with two phenyl rings,
including 4-phenylbenzene (11), 4-benzyloxybenzene (12) and 3-benzoylbenzene (13),
seemed to be favored for this position. Compound 12 was the most potent derivative
in this series, which had a nearly 4-fold improvement in the IC50 value as compared
to compound 1. The insertion of piperidine (14) or thiazole (15) between phenyl and
carbonyl resulted in a decrease of activity. When the 3-indole groups (16-18) were
introduced at P4, the inhibitory activity against the CT-L activity was generally main-
tained. Interestingly, although the replacement of the indole ring with benzofuran
(19) resulted in a loss of inhibitory activity, further change of the furan ring into
c-lactone (20) significantly restored the activity. The incorporation of tyrosine-derived
moiety at P4 (21 and 22) led to a reduction of the activity. The structural variation
on P4 suggested that two aromatic rings with appropriate distance and orientation

442
Y.-J. YANG ET AL.
might be important for the preservation of inhibitory activities against the CT-L
activity of human 20S proteasome. This is in consistent with the previous docking
results, which underscored p-p stacking and p-cation interactions with residues Tyr
96 and His 98, respectively [21].
Different amino acids were then introduced at P3 (23-28) with the P4 and P1 moi-
eties of compound 1 intact. However, all the amino acids except valine (25) resulted
in significant decline in activity. It was suggested that the asparagine residue might be
optimal for the P3 area.
According to previous docking studies, the P1 area of compound 1 forms a hydro-
gen bond network with Gly47 and Thr1 [21]. Consistently, compounds 29-31, which
were deprived of the hydrogen bond forming atoms, showed reduced inhibitory activ-
ity against CT-L activity. Instead, when L-allo-Thr-OBn was introduced at P1, the
inhibitory activity significantly varied with different substituents at P4 (32–36), and a
biphenyl group was obviously favored at P4 (32). D-Thr-OMe and D-Ser-OMe were
also introduced at P1 (37 and 38), however, such a structural variation led to a com-
plete loss in activity.
The nine most active compounds (11-13, 16-18, 20, 25 and 32) were further eval-
uated for their inhibition on T-L and C-L activities of human 20S proteasome. As
shown in Table 1, all these compounds selectively inhibited the CT-L activity, while
displayed no significant inhibition against the T-L and C-L activities.
2.3. Molecular modeling
Compound 18 was selected as a representative compound for molecular docking to
investigate the interaction mode of this compound class with the b5 subunit of the
20S proteasome. As depicted in Figure 2, compound 18 could situate in the active
site of the 20S proteasome with the 3-indole group extending to the hydrophobic
region of the active site. Extensive hydrogen bonds were monitored between com-
pound 18 and residues Gly47, Thr1, Ala49, Thr21 and Ala50 (b5) and Asp114 (b6).
Based on the interaction modes of non-covalent 20S proteasome inhibitors TMC-95A
and compound 1, we designed and synthesized a series of linear dipeptides (8–38) via a
fragment-based strategy. Most compounds exhibited moderate to potent inhibition
against the CT-L activity of 20S proteasome. Among them, nine compounds showed sig-
nificant inhibitory activities with IC50 values at submicromolar levels, and two most active
compounds, 12 and 18, had IC50 values of 0.17 and 0.20 lM, respectively. Furthermore,
no obvious inhibition against T-L and C-L activities was observed for nine most active
compounds. Molecular docking on compound 18 revealed the interaction modes of this
compound class. The results suggested that these dipeptides might represent potential
leads for the development of novel 20S proteasome inhibitors.
3. Experimental
3.1. Chemistry
All chemical reagents and solvents were obtained commercially and were used with-
out further purification. An SGW X-4A microscopic apparatus was used to determine

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
443
Figure 2. The binding mode of compound 18 proposed by docking study. (a) 2 D-plots: Residues
from b5 and b6 subunits of the 20S proteasome were represented with letters K and L, respect-
ively. Hydrogen bonding and p-cation interactions were shown in purple and green, respectively.
(b) 3 D-plots: Hydrogen bonding interactions were shown as green dotted lines.
1
melting points which are uncorrected. H-NMR spectra were recorded on Varian
Mercury 300 or 400 MHz spectrometers with tetramethylsilane (TMS) as internal
standard. Mass spectra with HR-ESI mode were recorded on a LC/MSD TOF instru-
ment from Agilent Technologies.
Compounds 4-38 were synthesized with reported methods [9]. Briefly, 2
(11 mmol), 3 (10 mmol), HATU (12 mmol), DMF (50 ml) and DIEA (36 mmol) were
added to a 200 ml round bottom flask. The mixture was stirred overnight at room
temperature. After removal of the solvent under reduced pressure, 100 ml ethyl acet-
ate was added and the organic phase was washed by water, saturated sodium hydro-
carbonate and brine sequentially. The removal of the solvent gave 4 which were used
in the next step without further purification. 4 were suspended with 10% Pd/C
(100 mg) in methanol (40 ml) under hydrogen atmosphere. The mixture was stirred
overnight at room temperature. After filtration, the filtrate was concentrated to give
5. Then, DMF (30 ml), amine (9 mmol), HATU (12 mmol) and DIEA (36 mmol) were
added to 5. The mixture was stirred overnight at room temperature. After the same
process for 4, 6 was afforded by column chromatography. 6 (5 mmol) was dissolved
in TFA/DCM (10 ml/10 ml), the mixture was stirred for 4 hours at room temperature.
Then the solvent was removed to give 7 without further purification. 7 (0.5 mmol),
acids (0.5 mmol), HATU (0.6 mmol) and DIEA (1.8 mmol) were dissolved in DMF

444
Y.-J. YANG ET AL.
(10 ml), and the mixture was then stirred overnight at room temperature. The reac-
tion mixture was then concentrated, and the desired compounds 8–38 were obtained
by column chromatography.
3.1.1. N-2-(4-Methoxyphenyl) acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (8)
1
Yield: 71.7%; mp 217-218 ꢁC; H-NMR (400 MHz, DMSO-d6): d 9.63 (s, 1H), 8.38 (d,
J ¼ 7.6 Hz, 2H), 7.67 (s, J ¼ 8.1 Hz, 1H), 7.43 (s, 1H), 7.16 (d, J ¼ 7.7 Hz, 2H), 6.97 (d,
J ¼ 8.2 Hz, 1H), 6.91 (s, 1H), 6.82 (d, J ¼ 7.9 Hz, 2H), 6.39(s, 1H), 6.32 (d, J ¼ 8.1 Hz,
1H), 4.58-4.63 (m, 1H), 4.11-4.15 (m, 4H), 3.71 (s, 3H, OCH3), 3.66 (s, 3H, OCH3),
3.38 (s, 2H), 2.54-2.58 (m, 1H), 1.95-2.00 (m, 1H), 0.70-0.75 (m, 6H, C(CH3)2); HR-
ESI-MS: m/z 515.2491 [M þ H]þ (calcd for C26H35N4O7, 515.2506).
3.1.2. N-2-(4-Trifluoromethylphenyl)acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (9)
1
Yield: 36.6%; mp 227-228 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.65 (s, 1H), 8.63 (d,
J ¼ 8.1 Hz, 1H), 8.35 (brs, 1H), 7.59-7.69 (m, 3H), 7.46-7.48 (m, 3H), 6.85-7.01 (m,
2H), 6.39 (s, 1H), 6.29 (d, J ¼ 7.6 Hz, 1H), 4.60 (d, J ¼ 7.0 Hz, 1H), 4.09 (brs, 3H),
3.64 (s, 3H, OCH3), 3.57 (s, 2H), 2.54-2.59 (m, 1H), 2.38-2.43 (m, 1H), 1.91-1.97 (m,
1H), 0.65-0.71 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 553.2247 [M þ H]þ (calcd for
C26H32F3N4O6, 553.2274).
3.1.3. N-2-(1-Naphthyl)acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (10)
1
Yield: 42.6%; mp 235-236 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.54 (s, 1H), 8.52 (d,
J ¼ 7.2 Hz, 1H), 8.32-8.34 (m, 1H), 8.03-8.07 (m, 1H), 7.87-7.90 (m, 1H), 7.77-7.80
(m, 1H), 7.70 (d, J ¼ 7.2 Hz, 1H), 7.47-7.50 (m, 2H), 7.40-7.41 (m, 3H), 6.92-6.97
(m, 2H), 6.29-6.34 (m, 2H), 4.61-4.67 (m, 1H), 4.09-4.16 (m, 3H), 3.93 (s, 2H), 3.64
(s, 3H, OCH3), 2.54-2.61 (m, 1H), 2.41-2.47 (m, 1H), 1.91-1.97 (m, 1H), 0.65-
0.72 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 535.2568 [M þ H]þ (calcd for
C29H35N4O6, 535.2557).
3.1.4. N-2-(1-Biphenyl)acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (11)
1
Yield: 43.6%; mp 231-232 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.57 (s, 1H), 8.44 (d,
J ¼ 6.7 Hz, 1H), 8.34 (s, 1H), 7.50-7.75 (m, 5H), 7.40-7.46 (m, 3H), 7.32-7.35 (m, 3H),
6.93-6.98 (m, 2H), 6.29-6.34 (m, 2H), 4.63 (d, J ¼ 6.9 Hz, 1H), 4.12 (brs, 3H), 3.65 (s,
3H, OCH3), 3.50 (s, 2H), 2.55-2.62 (m, 1H), 2.36-2.44 (m, 1H), 1.97 (d, J ¼ 6.5 Hz,
1H), 0.70-0.75 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 561.2697 [M þ H]þ (calcd for
C31H37N4O6, 561.2713).
3.1.5. N-2-(4-Benzyloxyphenyl)acyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (12)
1
Yield: 54.2%; mp 217-218 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.57 (s, 1H), 8.54 (d,
J ¼ 7.4 Hz, 1H), 8.34-8.36 (m, 1H), 7.82 (d, J ¼ 8.7 Hz, 2H), 7.73 (d, J ¼ 8.8 Hz, 1H),
7.34-7.48 (m, 6H), 7.09 (d, J ¼ 8.7 Hz, 2H), 6.98 (d, J ¼ 8.2 Hz, 2H), 6.92 (s, 1H),
6.32-6.36 (m, 2H), 5.18 (s, 2H), 4.77-4.83 (m, 1H), 4.11-4.21 (m, 3H), 3.67 (s, 3H,
OCH3), 2.56-2.69 (m, 2H), 1.95-2.02 (m, 1H), 0.75-0.81 (m, 6H, C(CH3)2); HR-ESI-
MS: m/z 577.2662 [M þ H]þ (calcd for C31H37N4O7, 577.2662).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
445
3.1.6.
N-2-(4-Benzoylphenyl)acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxyben-
zyl) (13)
1
Yield: 64.4%; mp 197-199 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.53 (s, 1H), 8.46 (d,
J ¼ 7.9 Hz, 1H), 8.30-8.34 (m, 1H), 7.64-7.73 (m, 5H), 7.52-7.57 (m, 4H), 7.45 (t,
J ¼ 7.6 Hz, 1H), 7.37 (s, 1H), 6.95 (d, J ¼ 8.2 Hz, 1H), 6.91 (s, 1H), 6.29-6.34 (m, 2H),
4.58-4.65 (m, 1H), 4.08-4.13 (m, 3H), 3.64 (s, 3H, OCH3), 3.55 (s, 2H), 2.52-2.60 (m,
1H), 2.33-2.40 (m, 1H), 1.90-1.97 (m, 1H), 0.64-0.69 (m, 6H, C(CH3)2); HR-ESI-MS:
m/z 589.2642 [M þ H]þ (calcd for C32H37N4O7, 589.2662).
3.1.7.
N-4-(4-Fluorobenzylpiperidinyl)acyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxy-
benzyl) (14)
1
Yield: 50%; mp 224-225 ꢁC. H-NMR (300 MHz, DMSO-d6): d 9.89 (s, 1H), 8.20-8.32
(m, 2H), 7.63-7.74 (m, 2H), 7.29-7.40 (m, 2H), 7.08-7.12 (m, 2H), 6.86-6.91 (m, 2H),
6.22-6.33 (m, 2H), 4.53-4.57 (m, 1H), 4.02-4.10 (m, 3H), 3.63 (s, 3H, OCH3), 3.39 (s,
2H), 2.74-2.77 (m, 1H), 2.37-2.55 (m, 2H), 1.82-1.97 (m, 5H), 1.50-1.60 (m, 4H),
0.76-0.78 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 586.3036 [M þ H]þ (calcd for
C30H41FN5O6, 586.3041).
3.1.8. N-2-(5-Methyl-2-phenylthiazolyl)acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxy-
benzyl) (15)
1
Yield: 50.4%; mp 215-216 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.69 (s, 1H), 8.44 (d,
J ¼ 7.5 Hz, 1H), 8.34-8.38 (m, 1H), 7.83-7.85 (m, 2H), 7.69 (d, J ¼ 8.7 Hz, 1H), 7.44-
7.46 (m, 4H), 6.94-6.98 (m, 2H), 6.36 (s, 1H), 6.32 (d, J ¼ 8.3 Hz, 1H), 4.61-4.68 (m,
1H), 4.10-4.17 (m, 3H), 3.64-3.71 (m, 2H), 3.66 (s, 3H, OCH3), 2.55-2.62 (m, 1H),
2.43-2.50 (m, 1H), 2.41 (s, 3H), 1.94-2.00 (m, 1H), 0.70-0.75 (m, 6H, C(CH3)2); HR-
ESI-MS: m/z 582.2373 [M þ H]þ (calcd for C29H36N5O6S, 582.2386).
3.1.9. N-2-(3-Indolyl)acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (16)
1
Yield: 63%; mp 214-215 ꢁC; H-NMR (300 MHz, CD3OD): d 8.27 (s, 1H), 8.02 (d,
J ¼ 7.8 Hz, 1H), 7.48 (d, J ¼ 9.0 Hz, 1H), 7.27 (d, J ¼ 9.0 Hz, 1H), 7.14 (s, 1H), 6.92-
7.06 (m, 3H), 6.28-6.30 (m, 2H), 4.72 (t, J ¼ 6.0 Hz, 1H), 4.18 (s, 2H), 4.01-4.08 (m,
1H), 3.70 (s, 3H, OCH3), 3.65-3.69 (m, 2H), 2.51-2.67 (m, 3H), 0.66-0.72 (m, 6H,
C(CH3)2); HR-ESI-MS: m/z 524.2512 [M þ H]þ (calcd for C27H34N5O6, 524.2509).
3.1.10. N-2-(3-(5-Methoxyindolyl))acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxyben-
zyl) (17)
1
Yield: 35.7%; mp 206-208 ꢁC; H-NMR (300 MHz, DMSO-d6): d 10.70 (s, 1H), 9.57
(s, 1H), 8.36 (s, 1H), 8.28 (d, J ¼ 7.3 Hz, 1H), 7.73 (d, J ¼ 8.8 Hz, 1H), 7.44 (s, 1H),
7.20 (d, J ¼ 8.8 Hz, 1H), 7.14 (s, 1H), 7.03 (s, 1H), 6.96 (d, J ¼ 8.1 Hz, 1H), 6.90 (s,
1H), 6.69 (d, J ¼ 6.9 Hz, 1H), 6.35 (s, 1H), 6.30 (d, J ¼ 8.2 Hz, 1H), 4.61-4.65 (m, 1H),
4.12 (brs, 3H), 3.75 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 3.51 (s, 2H), 2.50-2.60 (m,
1H), 2.37-2.45 (m, 1H), 1.93-1.98 (m, 1H), 0.68-0.73 (m, 6H, C(CH3)2); HR-ESI-MS:
m/z 554.2605 [M þ H]þ (calcd for C28H36N5O7, 554.2615).

446
Y.-J. YANG ET AL.
3.1.11.
N-2-(3-Indolyl)-2-ketone-acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxyben-
zyl) (18)
1
Yield: 50.2%; mp 215-216 ꢁC; H-NMR (300 MHz, DMSO-d6): d 8.91 (d, J ¼ 7.9 Hz,
1H), 8.73 (s, 1H), 8.37 (t, J ¼ 5.6 Hz, 1H), 8.22-8.25 (m, 1H), 7.87 (d, J ¼ 8.6 Hz, 1H),
7.53-7.56 (m, 1H), 7.43 (s, 1H), 7.25-7.28 (m, 2H), 6.98 (d, J ¼ 8.2 Hz, 2H), 6.38 (d,
J ¼ 2.2 Hz, 1H), 6.31 (dd, J ¼ 8.4 Hz, 2.2 Hz, 1H), 4.68-4.75 (m, 1H), 4.09-4.21 (m,
3H), 3.63 (s, 3H, OCH3), 3.33 (s, 2H), 2.60-2.67 (m, 2H), 1.94-2.02 (m, 1H), 0.79-
0.81 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 538.2294 [M þ H]þ (calcd for
C27H32N5O7, 538.2302).
3.1.12. N-2-(6-Methoxybenzofuranyl)acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxy-
benzyl) (19)
1
Yield: 40%; mp 204-206 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.54 (s, 1H), 8.40-8.46
(m, 1H), 8.28-8.34 (m, 1H), 7.69-7.73 (m, 1H), 7.67 (s, 1H), 7.47 (d, J ¼ 9.0 Hz, 1H),
7.36-7.39 (m,1H), 7.11-7.12 (m, 1H), 6.92-6.98 (m, 2H), 6.80-6.84 (m, 1H), 6.26-6.34
(m, 2H), 4.62-4.66 (m, 1H), 4.09-4.16 (m, 3H), 3.76 (s, 3H, OCH3), 3.64 (s, 3H,
OCH3), 3.50 (s, 2H), 2.52-2.59 (m, 1H), 2.37-2.44 (m, 1H), 1.93-1.99 (m, 1H), 0.68-
0.77 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 555.2458 [M þ H]þ (calcd for
C28H35N4O8, 555.2455).
3.1.13.
N-2-(3-Isobenzofuran-1-one)acetyl-L-Asn-L-Val-NH-(2-hydroxy-4-methoxy-
benzyl) (20)
1
Yield: 30%; mp 262-263 ꢁC. H-NMR (300 MHz, DMSO-d6): d 9.55 (d, J ¼ 2.0 Hz,
1H), 8.35-8.41 (m, 2H), 7.81 (d, J ¼ 7.6 Hz, 1H), 7.74 (d, J ¼ 7.5 Hz, 1H), 7.65-7.70
(m, 1H), 7.58 (t, J ¼ 7.6 Hz, 1H), 7.43 (d, J ¼ 9.0 Hz, 1H), 6.96-7.01 (m, 2H), 6.31-6.36
(m, 2H), 5.86-5.92 (m, 1H), 4.71-4.75 (m, 1H), 4.12-4.16 (m, 3H), 3.73(m, 1H), 3.66
(s, 3H, OCH3), 2.81-2.88 (m, 1H), 2.67-2.75 (m, 1H), 2.54-2.62 (m, 1H), 2.37-2.45
(m, 1H), 2.03-2.09 (m, 1H), 0.83-0.85 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 541.2307
[M þ H]þ (calcd for C27H33N4O8, 541.2298).
3.1.14. N-(tert-butyl (S)-(1-(4-Hydroxyphenyl)-3-oxobutanyl))acyl-L-Asn-L-Val-NH-(2-
hydroxy-4-methoxybenzyl) (21)
1
Yield: 64.7%; mp 162-163 ꢁC; H-NMR (400 MHz, DMSO-d6): d 9.53 (s, 1H), 9.11 (s,
1H), 8.32-8.34 (m, 1H), 8.19 (d, J ¼ 7.5 Hz, 1H), 7.71 (d, J ¼ 8.6 Hz, 1H), 7.40 (s, 1H),
6.94-7.02 (m, 4H), 6.81 (d, J ¼ 8.5 Hz, 1H), 6.61 (d, J ¼ 8.3 Hz, 2H), 6.34 (d,
J ¼ 2.3 Hz, 1H), 6.30 (dd, J ¼ 8.3 Hz, 2.3 Hz, 1H), 4.57-4.63 (m, 1H), 4.10-4.17 (m,
4H), 3.64 (s, 3H, OCH3), 2.80-2.83 (m, 1H), 2.54-2.60 (m, 2H), 2.41-2.45 (m, 1H),
1.97-2.02 (m, 1H), 1.28 (s, 9H), 0.77-0.80 (m, 6H, C(CH3)2); HR-ESI-MS: m/z
630.3111 [M þ H]þ (calcd for C31H44N5O9, 630.3139).
3.1.15. N-((S)-N-((S)-1-(4-Hydroxyphenyl)-3-oxobutan-2-yl))acyl-L-Asn-L-Val-NH-(2-
hydroxy-4-methoxybenzyl) (22)
1
Yield: 13.7%; mp 220-221 ꢁC; H-NMR (300 MHz, CD3OD): d 6.96-7.02 (m, 3H), 6.62
(d, J ¼ 8.4 Hz, 2H), 6.26-6.31 (m, 2H), 4.62-4.67 (m, 1H), 4.48-4.53 (m, 1H), 4.21 (s,
2H), 4.13 (d, J ¼ 6.0 Hz, 1H), 3.66 (s, 3H, OCH3), 2.98-3.02 (m, 1H), 2.62-2.75 (m,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
447
3H), 2.04-2.18 (m, 2H), 1.13-1.30 (m, 2H), 0.98 (d, J ¼ 6.0 Hz, 2H), 0.82-0.86 (m, 6H,
C(CH3)2), 0.72-0.76 (m, 3H), 0.54-0.69 (m, 1H); HR-ESI-MS: m/z 614.3188 [M þ H]þ
(calcd for C31H44N5O8, 614.3190).
3.1.16.
N-2-(3-Phenoxyphenyl)acetyl-L-Ser-L-Val-NH-(2-hydroxy-4-methoxyben-
zyl) (23)
1
Yield: 52.6%; mp 145-146 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.54 (s, 1H), 8.18-
8.25 (m, 2H), 7.73 (d, J ¼ 8.8 Hz, 1H), 7.36 (t, J ¼ 7.5 Hz, 2H), 7.27 (t, J ¼ 7.5 Hz, 1H),
7.11 (t, J ¼ 7.5 Hz, 1H), 6.94-7.03 (m, 5H), 6.82 (dd, J ¼ 7.8 Hz, 1.9 Hz, 1H), 6.34 (d,
J ¼ 2.3 Hz, 1H), 6.30 (dd, J ¼ 8.3 Hz, 2.4 Hz, 1H), 4.99 (t, J ¼ 5.1 Hz, 1H), 4.35-4.41
(m, 1H), 4.08-4.19 (m, 3H), 3.64 (s, 3H, OCH3), 3.50-3.55 (m, 2H), 3.48 (s, 2H),
1.93-2.0 (m, 1H), 0.74-0.78 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 550.2549 [M þ H]þ
(calcd for C30H36N3O7, 550.2553).
3.1.17.
N-2-(3-Phenoxyphenyl)acetyl-L-Thr-L-Val-NH-(2-hydroxy-4-methoxyben-
zyl) (24)
1
Yield: 20%; mp 135-136 ꢁC; H-NMR (300 MHz, DMSO-d6): d 9.56 (s, 1H), 8.27-8.31
(m, 1H), 8.06 (d, J ¼ 8.4 Hz, 1H), 7.66 (d, J ¼ 8.4 Hz, 1H), 7.38 (t, J ¼ 8.0 Hz, 2H),
7.29 (t, J ¼ 8.0 Hz, 1H), 7.13 (t, J ¼ 7.4 Hz, 1H), 7.06 (d, J ¼ 7.8 Hz, 1H), 6.97-7.01 (m,
4H), 6.85 (d, J ¼ 7.8 Hz, 1H), 6.36 (d, J ¼ 2.4 Hz, 1H), 6.31 (dd, J ¼ 8.3 Hz, 2.4 Hz,
1H), 4.95 (d, J ¼ 4.8 Hz, 1H), 4.35-4.41 (m, 1H), 4.28-4.32 (m, 1H), 4.1-4.2 (m, 2H),
3.92-3.98 (m, 1H), 3.66 (s, 3H, OCH3), 3.52-3.56 (m, 2H), 1.91-2.00 (m, 1H), 0.95 (d,
J ¼ 6.0 Hz, 3H), 0.78-0.82 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 564.2716 [M þ H]þ
(calcd for C31H38N3O7, 564.2710).
3.1.18.
N-2-(3-Phenoxyphenyl)acetyl-L-Val-L-Val-NH-(2-hydroxy-4-methoxyben-
zyl) (25)
1
Yield: 22%; mp 140-141 ꢁC; H-NMR (300 MHz, CD3OD): d 7.24-7.35 (m, 3H), 7.02-
7.10 (m, 3H), 6.94-6.99 (m, 3H), 6.85 (d, J ¼ 8.1 Hz, 1H), 6.31-6.35 (m, 2H), 4.24(d,
J ¼ 6.0 Hz, 2H), 4.08-4.19 (m, 2H), 3.71 (s, 3H, OCH3), 3.52 (d, J ¼ 6.0 Hz, 2H), 1.96-
2.05 (m, 2H), 0.84-0.87 (m, 12H, C(CH3)2); HR-ESI-MS: m/z 562.2902 [M þ H]þ
(calcd for C32H40N3O6, 562.2917).
3.1.19. N-2-(3-Indolyl)acetyl-L-Ser-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (26)
1
Yield: 59%; mp 198-200 ꢁC; H-NMR (300 MHz, CD3OD): d 7.56 (d, J ¼ 8.1 Hz, 1H),
7.34 (d, J ¼ 8.1 Hz, 1H), 7.20 (s, 1H), 7.12-7.00 (m, 3H), 6.31-6.35 (m, 2H), 4.45-4.52
(m, 1H), 4.15-4.24 (m, 3H), 3.75-3.81 (m, 1H), 3.73 (s, 2H), 3.67 (s, 3H, OCH3), 2.69
(m, 1H), 1.98-2.04 (m,1H), 0.77-0.84 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 497.2403
[M þ H]þ (calcd for C26H33N4O6, 497.2400).
3.1.20. N-2-(3-Indolyl)acetyl-L-Thr-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (27)
1
Yield: 49%; mp 94-95 ꢁC; H-NMR (300 MHz, DMSO-d6): d 10.84 (brs, 1H), 9.54 (s,
1H), 8.25-8.29 (m, 1H), 7.77 (d, J ¼ 7.9 Hz, 1H), 7.64(d, J ¼ 8.7 Hz, 1H), 7.53 (d,
J ¼ 7.8 Hz, 1H), 7.31 (d, J ¼ 8.1 Hz, 1H), 7.20 (s, 1H), 7.04 (t, J ¼ 7.5 Hz, 1H), 6.91-
6.98 (m, 2H), 6.34 (d, J ¼ 2.3 Hz, 1H), 6.30 (dd, J ¼ 8.4 Hz, 2.2 Hz, 1H), 4.92 (d,

448
Y.-J. YANG ET AL.
J ¼ 4.8 Hz, 1H), 4.28-4.32 (m, 1H), 4.08-4.20 (m, 3H), 3.92-3.97 (m, 1H), 3.60-3.64 (m,
2H), 3.55 (s, 3H, OCH3), 1.91-1.97 (m, 1H), 0.95 (d, J ¼ 6.0 Hz, 3H), 0.76-0.80 (m, 6H,
C(CH3)2); HR-ESI-MS: m/z 511.2555 [Mþ H]þ (calcd for C27H35N4O6, 511.2557).
3.1.21. N-2-(3-Indolyl)acetyl-L-Val-L-Val-NH-(2-hydroxy-4-methoxybenzyl) (28)
1
Yield: 29%; mp 145-146 ꢁC; H-NMR (300 MHz, DMSO-d6): d 10.81 (brs, 1H), 9.54
(s, 1H), 8.19-8.23 (m, 1H), 7.87 (d, J ¼ 8.9 Hz, 1H), 7.80 (d, J ¼ 8.8 Hz, 1H), 7.52 (d,
J ¼ 7.9 Hz, 1H), 7.30 (d, J ¼ 8.1 Hz 1H), 7.16 (s, 1H), 6.89-7.05 (m, 3H), 6.34 (d,
J ¼ 2.3 Hz, 1H), 6.28 (dd, J ¼ 8.3 Hz, 2.3 Hz, 1H), 4.18-4.23 (m, 1H), 4.09-4.14 (m,
3H), 3.64 (s, 3H, OCH3), 3.50-3.55 (m, 2H), 1.86-1.97 (m, 2H), 0.74-0.78 (m, 12H,
C(CH3)2); HR-ESI-MS: m/z 509.2762 [M þ H]þ (calcd for C28H37N4O5, 509.2764).
3.1.22. N-2-(3-Phenoxyphenyl)acetyl-L-Asn-L-Val-NH-(2-chlorobenzyl) (29)
1
Yield: 73.4%; mp 252-253 ꢁC; H-NMR (300 MHz, DMSO-d6): d 8.52 (t, J ¼ 5.8 Hz,
1H), 8.41 (d, J ¼ 7.5 Hz, 1H), 7.81 (d, J ¼ 8.1 Hz, 1H), 7.36-7.41 (m, 4H), 7.25-7.32
(m, 4H), 7.13 (t, J ¼ 7.2 Hz, 1H), 6.95-7.05 (m, 5H), 6.84 (dd, J ¼ 8.1 Hz, 1.8 Hz, 1H),
4.62-4.68 (m, 1H), 4.31-4.33 (m, 2H), 4.14-4.19 (m, 1H), 3.46 (s, 2H), 2.55-2.62 (m,
1H), 2.36-2.43 (m, 1H), 2.01-2.08 (m, 1H), 0.74-0.80 (m, 6H, C(CH3)2); HR-ESI-MS:
m/z 565.2232 [M þ H]þ (calcd for C30H34ClN4O5, 565.2218).
3.1.23. N-2-(3-Phenoxyphenyl)acetyl-L-Asn-L-Val-NH-(4-methylbenzyl) (30)
1
Yield: 79%; mp 249-250 ꢁC; H-NMR (300 MHz, DMSO-d6): d 8.36-8.41 (m, 2H),
7.67 (d, J ¼ 8.7 Hz, 1H), 7.34-7.38 (m, 3H), 7.26 (t, J ¼ 7.8 Hz, 1H), 7.09-7.13 (m, 5H),
6.93-7.02 (m, 5H), 6.82 (d, J ¼ 8.0 Hz, 1H), 4.58-4.64 (m, 1H), 4.07-4.19 (m, 3H), 3.44
(s, 2H), 2.52-2.59 (m, 1H), 2.32-2.40 (m, 1H), 2.24 (s, 3H, CH3), 1.96-2.00 (m, 1H),
0.68-0.78 (m, 6H, C(CH3)2); HR-ESI-MS: m/z 545.2761 [M þ H]þ (calcd for
C31H37N4O5, 545.2764).
3.1.24. N-2-(3-Phenoxyphenyl)acetyl-L-Asn-L-Val-NH-(2-thienyl) (31)
1
Yield: 73.2%; mp 240-241 ꢁC; H-NMR (300 MHz, DMSO-d6): d 8.51-8.58 (m, 1H),
8.32-8.39 (m, 1H), 7.67(d, J ¼ 8.9 Hz, 1H), 7.34-7.39 (m, 4H), 7.26 (t, J ¼ 7.5 Hz, 1H),
7.11 (t, J ¼ 7.5 Hz, 1H), 6.96-7.02 (m, 3H), 6.89-6.92 (m, 4H), 6.80-6.84 (m, 1H),
4.58-4.64 (m, 1H), 4.36-4.40 (m, 2H), 4.06-4.13 (m, 1H), 3.44 (s, 2H), 2.51-2.58
(m,1H), 2.32-2.40 (m, 1H), 1.96-2.00 (m, 1H), 0.69-0.78 (m, 6H, C(CH3)2); HR-ESI-
MS: m/z 537.2166 [M þ H]þ (calcd for C28H33N4O5S, 537.2172).
3.1.25. N-2-(1-Biphenyl)acetyl-L-Asn-L-Val-L-allo-Thr-OBn (32)
1
Yield: 30%; mp 242-243 ꢁC; H-NMR (300 MHz, DMSO-d6): d 8.44 (d, J ¼ 7.4 Hz,
1H), 8.19 (d, J ¼ 6.8 Hz, 1H), 7.62 (d, J ¼ 7.3 Hz, 4H), 7.55 (d, J ¼ 8.0 Hz, 4H), 7.44 (t,
J ¼ 7.3 Hz, 3H), 7.33 (t, J ¼ 7.4 Hz, 5H), 6.90 (s, 1H), 5.01 (d, J ¼ 5.1 Hz, 1H), 4.60-
4.63 (m, 1H), 4.14-4.26 (m, J ¼ 6.0 Hz, 2H), 3.85-3.90 (m, 1H), 3.58 (s, 3H), 3.50 (s,
2H), 2.53-2.62 (m, 1H), 2.34-2.42 (m, 1H), 1.88-1.93 (m, 1H), 1.09 (d, J ¼ 6.0 Hz, 3H,
CH3), 0.76 (d, J ¼ 6.3 Hz, 3H, CH3), 0.69 (d, J ¼ 6.4 Hz, 3H, CH3); HR-ESI-MS: m/z
617.2977 [M þ H]þ (calcd for C34H41N4O7, 617.2975).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
449
3.1.26. N-2-(3-Indolyl)acetyl-L-Asn-L-Val-L-allo-Thr-OBn (33)
1
Yield: 44%; mp 234-235 ꢁC; H-NMR (300 MHz, DMSO-d6): d 10.85 (brs, 1H), 8.26
(d, J ¼ 7.7 Hz, 2H), 7.51-7.58 (m, 2H), 7.34 (brs, 7H), 7.19 (s, 1H), 7.05 (t, J ¼ 7.2 Hz,
1H), 6.93 (t, J ¼ 7.2 Hz, 1H), 6.87 (s, 1H), 5.10 (s, 2H), 5.06 (d, J ¼ 5.5 Hz, 1H), 4.54-
4.71 (m, 1H), 4.24 (t, J ¼ 7.0 Hz, 2H), 3.90-3.96 (m, 1H), 3.55 (s, 2H), 2.50-2.58 (m,
1H), 2.34-2.42 (m, 1H), 1.82-1.92 (m, 1H), 1.10 (d, J ¼ 6.3 Hz, 3H, CH3), 0.72 (d,
J ¼ 6.7 Hz, 3H, CH3), 0.65 (d, J ¼ 6.7 Hz, 3H, CH3); HR-ESI-MS: m/z 580.2732
[M þ H]þ (calcd for C30H38N5O7, 580.2771).
3.1.27. N-2-(4-Cyclohexylphenyl)acyl-L-Asn-L-Val-L-allo-Thr-OBn (34)
1
Yield: 43%; mp 219-220 ꢁC; H-NMR (300 MHz, CD3OD): d 7.77-7.82 (m, 2H), 7.33-
7.37 (m, 7H), 5.19 (s, 2H), 4.94-4.97 (m, 1H), 4.46-4.48 (m, 1H), 4.30-4.32 (m, 1H),
4.11-4.13 (m, 1H), 2.77-2.83 (m, 2H), 2.54-2.60 (m, 1H), 2.07-2.09 (m, 1H), 1.75-1.85
(m, 5H), 1.42-1.47 (m, 3H), 1.20-1.23 (m, 3H), 0.88-0.90 (m, 6H, C(CH3)2); HR-ESI-
MS: m/z 609.3225 [M þ H]þ (calcd for C33H45N4O7, 609.3288).
3.1.28. N-2-(3,4,5-Trimethoxylphenyl)propionyl -L-Asn-L-Val-L-allo-Thr-OBn (35)
1
Yield: 51.7%; mp 241-242 ꢁC; H-NMR (300 MHz, DMSO-d6): d 8.35 (d, J ¼ 7.3 Hz,
1H), 8.27 (d, J ¼ 7.8 Hz, 1H), 7.62 (d, J ¼ 9.2 Hz, 1H), 7.42 (s, 1H), 6.89 (s, 5H), 6.88
(s, 1H), 6.51 (s, 2H), 5.14 (d, J ¼ 5.5 Hz, 1H), 5.10 (s, 2H), 4.60-4.64 (m, 1H), 4.22-
4.28 (m, 2H), 3.92-3.98 (m, 1H), 3.74 (s, 6H, OCH3), 3.61 (s, 3H, OCH3), 2.71-2.76
(m, 2H), 2.36-2.45 (m, 4H), 1.92-1.98 (m, 1H), 1.11 (d, J ¼ 6.0 Hz, 3H), 0.79 (d,
J ¼ 6.0 Hz, 3H, CH3), 0.74 (d, J ¼ 6.0 Hz, 3H, CH3); HR-ESI-MS: m/z 645.3112
[M þ H]þ (calcd for C32H45N4O10, 645.3136).
3.1.29. N-2-(2-Pyrazinyl)acyl-L-Asn-L-Val-L-allo-Thr-OBn (36)
1
Yield: 51.5%; mp 205-206 ꢁC; H-NMR (300 MHz, CD3OD): d 9.23 (s, 1H), 8.79 (s,
1H), 8.69 (s, 1H), 7.31-7.36 (m, 5H), 5.16 (s, 2H), 4.96-5.00 (m, 1H), 4.44 (d,
J ¼ 6.0 Hz, 1H), 4.27 (d, J ¼ 6.0 Hz, 1H), 4.08 (t, J ¼ 6.0 Hz, 1H), 2.83 (t, J ¼ 6.0 Hz,
2H), 2.05-2.11 (m, 4H), 1.21(m, 4H), 0.89 (m, 8H); HR-ESI-MS: m/z 529.2405
[M þ H]þ (calcd for C25H33N6O7, 529.2411).
3.1.30. N-2-(3-Phenoxyphenyl)acetyl-L-Asn-L-Val-D-allo-Thr-OMe (37)
1
Yield: 71.4%; mp 215-217 ꢁC; H-NMR (300 MHz, DMSO-d6): d 8.38 (d, J ¼ 7.2 Hz,
1H), 8.00 (d, J ¼ 7.2 Hz, 1H), 7.59 (d, J ¼ 9.0 Hz, 1H), 7.34-7.39 (m, 3H), 7.26 (t,
J ¼ 7.5 Hz, 1H), 7.11 (t, J ¼ 7.2 Hz, 1H), 6.96-7.02 (m, 2H), 6.88-6.92 (m, 2H), 6.81-
6.83 (m, 2H), 4.93-4.95 (m, 1H), 4.55-4.62 (m, 2H), 4.20-4.25 (m, 2H), 4.04-4.12 (m,
1H), 3.59 (s, 3H, OCH3), 3.43 (s, 2H), 2.52-2.60 (m, 2H), 2.30-2.38 (m, 1H), 1.94-
1.99 (m, 1H), 1.04 (d, J ¼ 6.3 Hz, 1H), 0.77 (d, J ¼ 6.8 Hz, 3H, CH3), 0.70 (d,
J ¼ 6.8 Hz, 3H, CH3); HR-ESI-MS: m/z 557.2605 [M þ H]þ (calcd for
C28H37N4O8, 557.2611).
3.1.31. N-2-(3-Phenoxyphenyl)acetyl-L-Asn-L-Val-D-Ser-OMe (38)
1
Yield: 58%; mp 165-166 ꢁC; H-NMR (300 MHz, DMSO-d6): d 8.41 (d, J ¼ 8.1 Hz,
1H), 8.28 (d, J ¼ 7.2 Hz, 1H), 7.55 (d, J ¼ 9.0 Hz, 1H), 7.36-7.41 (m, 3H), 7.28 (t,

450
Y.-J. YANG ET AL.
J ¼ 7.8 Hz, 1H), 7.13 (t, J ¼ 7.2 Hz, 1H), 6.98-7.04 (m, 2H), 6.90-6.94 (m, 2H), 6.82-
6.85 (m, 2H), 4.56-4.62 (m, 1H), 4.23-4.30 (m, 2H), 3.63-3.69 (m, 2H), 3.61 (s, 3H,
OCH3), 3.45 (s, 2H), 2.53-2.60 (m, 2H), 2.32-2.39 (m, 1H), 1.92-1.98 (m, 1H), 0.73
(d, J ¼ 6.8 Hz, 3H, CH3), 0.71 (d, J ¼ 6.8 Hz, 3H, CH3); HR-ESI-MS: m/z 543.2468
[M þ H]þ (calcd for C27H35N4O8, 543.2455).
3.2. Biological evaluation
The chymotrypsin-like, trypsin-like and caspase-like activities of the human 20S pro-
teasome activity were determined by monitoring the decrease in hydrolysis of the flu-
orogenic substrates Suc-LLVY-AMC, Bz-VGR-AMC and (Z)-LLE-bNA, respectively.
The rate of the cleavage reaction was measured with the excitation wavelength of
365 nm and the emission wavelength of 465 nm [14].
3.3. Molecular modeling
The structure of compound 18 was generated and molecular docking was performed
€
with Glide (version 2007, Schrodinger, LLC, New York). The structure of the 20S
proteasome (b5 and b6 subunits) was obtained from the Protein Data Bank (PDB
code: 3SDK). Prior to docking for 18, the co-crystal ligand was redocked into the
active site successfully within a RMSD value of 2.00 Å. All docking calculations were
run in the Standard Precision (SP) mode of Glide with default settings. The best
scored pose was kept with a SP docking score of ꢂ10.956 kcal/mol.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
We are grateful for the support from the National Natural Science Foundation of China
(21672263) and the CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-3-009).
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